The vertebrate retina is a relatively simple neuronal structure composed of five distinct types of neurons (photoreceptors, bipolar, horizontal, amacrine, and ganglion cells) and one class of glial (Muller) cells, arranged in well-defined layers. Several of the cell types have been morphologically and electrophysiologically characterized. In addition, many putative neurotransmitters have been identified in the retina, however their localization within specific cell types has been difficult to determine. We have developed a rapid method to chemically dissociate rat retinae and to separate large quantities of various cell types by centrifugation through a metrizamide density gradient. We demonstrate the separation of seven distinct bands (layers I through VII) of cells. Cells from layers II and IV were morphologically identified as red blood cells and photoreceptor cells, respectively. Other cell layers were biochemically unique in their content of key biosynthetic enzymes. Glutamate decarboxylase (GAD) (EC 4.1.1.15) is concentrated in layers I and VI, choline acetyltransferase (CAT) (EC 2.1.3.6) is present in layers VI and VII, and tyrosine hydroxylase (TH) (EC 1.14.16.2) is in layers I and VI. This method serves as the basis for separation and biochemical characterization of several retinal cell types.